Bordetella pertussis is the bacterial pathogen responsible for whooping cough in humans. Serotype markers for the bacterium have been defined by the ability of strain-specific polyclonal antisera to agglutinate the bacteria. E. K. Andersen first identified five distinctive agglutinogen factors in 1953 (Acta Pathol. Microbiol. Scand. 33:202-224 (1953)), and Eldering et al. subsequently added agglutinogen factor 6 (J. Bacteriol. 74:133-136 (1957)). Following whooping cough outbreaks, it has been noted that there tends to be a prevalence of certain Bordetella pertussis serotypes, and the serum agglutinin titers of human vaccinees appear to correlate with clinical protection from pertussis.
Some of the agglutinogen factors have been defined, for instance, the expression of lipooligosaccharide A (LOS A) by Bordetella pertussis cells appears to correlate with the presence of the serotype 1 agglutinogen factor. Likewise, serotype 2 and 6 agglutinogens have been found to correspond to fimbriae while the serotype 3 agglutinogen factor is composed of both fimbriae and a 69 kDa outer membrane protein.
The present inventors describe herein as an object of their invention, a new method for the isolation and purification of cell proteins, specifically the nonfimbrial 69 kDa outer membrane protein of Bordetella pertussis. The protein purified by the method of the present invention is useful in the preparation of Bordetella pertussis hybridomas and serotype-specific monoclonal antibodies (Mabs) thereto. The protein can also be advantageously utilized for a wide range of diagnostic, manufacturing and research purposes including, but not limited to inclusion in acellular pertussis vaccines.
Two groups have previously published purification procedures for the Bordetella pertussis 69 kDa protein or for the related 68 kDa protein from Bordetella pertussis, which is antigenically similar to the 69 kDa protein.
The most recently disclosed method is that described by Brennan et al. (Infect. Immun. 56:3189-3195 (1988)). In summary it is a multi-step procedure, the first step being the purification of the protein from the bacteria by heating the bacterial cells at 60.degree. C. for 1 hour. The resulting extract is then applied to a fetuin-Sepharose 4B column, followed by chromatography of the fractions containing the 69,000 Da protein on an immunoaffinity column in which a monoclonal antibody specific for the 69,000 Da protein was linked to agarose. The 69,000 Da protein was then eluted from the column with 6M urea.
The second purification procedure was disclosed by Novotny et al. (Infect. Immun. 50:199-206 (1985)) and involves as a first step the preparation of an acid glycine hydrolyzate of the bacteria. The resulting extract was dialyzed versus 0.025M Tris, pH 8.8, containing 0.035M NaCl, then chromatographed on DEAE-Trisacryl. Material which was not retained by the column was subjected to isoelectrofocusing. Pooled eluants from zones of pH 7.5 to 7.0 were applied to an immunoaffinity column in which a monoclonal antibody specific for the 68,000 Da Bordetella bronchiseptica protein was linked to Sepharose CL-6B. Sepharose CL is prepared by cross-linking agrose with 2,3-dibromopropanol and desulfating the resulting gel by alkaline hydrolysis under reducing conditions. The protein was eluted from the column with buffer containing 6M urea.
The invention described in the present application differs from the above purification schemes in that the procedures are substantially different and do not require the use of an immunoaffinity column and monoclonal antibodies which is advantageous, for the production of monoclonal antibodies is both time consuming and expensive. Therefore, this method is quicker and cheaper than methods previously disclosed.
In addition to the above, the process of the present invention also allows for easy scale-up. This is advantageous since large-scale purification processes are needed to produce sufficient quantities of proteins such as the 69,000 Da protein for use in vaccines. Similarly, the present process does not require or involve additional biologics (such as monoclonal antibodies), the use of which should be avoided, if possible, when producing proteins for inclusion in vaccines.
Further objects and advantages of the invention will become apparent from the following description.